Mechanism of subunit interaction at ketosynthase-dehydratase junctions in trans-AT polyketide synthases

Modular polyketide synthases (PKSs) produce numerous structurally complex natural products with diverse applications in medicine and agriculture. They typically consist of several multienzyme subunits that utilize structurally-defined docking domains (DDs) at their N- and C-termini to ensure correct assembly into functional multi-protein complexes. Here we report a fundamentally different mechanism for subunit assembly in trans-AT modular PKSs at the junction between ketosynthase (KS) and dehydratase (DH) domains. This involves direct interaction of a largely unstructured docking domain (DD) at the C-terminus of the KS with the surface of the downstream DH. Acyl transfer assays and mechanism-based cross-linking established that the DD is required for the KS to communicate with the acyl carrier protein appended to the DH. Two distinct regions for binding of the DD to the DH were identified using NMR spectroscopy, carbene foot-printing and mutagenesis, providing a foundation for future elucidation of the molecular basis for interaction specificity

Effects of the fungicide metiram in outdoor freshwater microcosms: responses of invertebrates, primary producers and microbes

The ecological impact of the dithiocarbamate fungicide metiram was studied in outdoor freshwater microcosms, consisting of 14 enclosures placed in an experimental ditch. The microcosms were treated three times (interval 7 days) with the formulated product BAS 222 28F (Polyram®). Intended metiram concentrations in the overlying water were 0, 4, 12, 36, 108 and 324 μg a.i./L. Responses of zooplankton, macroinvertebrates, phytoplankton, macrophytes, microbes and community metabolism endpoints were investigated. Dissipation half-life (DT50) of metiram was approximately 1–6 h in the water column of the microcosm test system and the metabolites formed were not persistent. Multivariate analysis indicated treatment-related effects on the zooplankton (NOECcommunity = 36 μg a.i./L). Consistent treatment-related effects on the phytoplankton and macroinvertebrate communities and on the sediment microbial community could not be demonstrated or were minor. There was no evidence that metiram affected the biomass, abundance or functioning of aquatic hyphomycetes on decomposing alder leaves. The most sensitive populations in the microcosms comprised representatives of Rotifera with a NOEC of 12 μg a.i./L on isolated sampling days and a NOEC of 36 μg a.i./L on consecutive samplings. At the highest treatment-level populations of Copepoda (zooplankton) and the blue-green alga Anabaena (phytoplankton) also showed a short-term decline on consecutive sampling days (NOEC = 108 μg a.i./L). Indirect effects in the form of short-term increases in the abundance of a few macroinvertebrate and several phytoplankton taxa were also observed. The overall community and population level no-observed-effect concentration (NOECmicrocosm) was 12–36 μg a.i./L. At higher treatment levels, including the test systems that received the highest dose, ecological recovery of affected measurement endpoints was fast (effect period < 8 weeks)

Flexible IgE epitope containing domains of Phl p 5 cause high allergenic activity.

This is the first study determining the three-dimensional structure of Phl p 5a which reveals a novel mechanism for high allergenic activity based on flexibly connected IgE-reactive domains which cross-link effector cell-bound IgE more efficiently than isolated rigid globular proteins. These findings may also form a basis for specific immunotherapy strategies

Evaluation of the Safety and Immunogenicity in Rhesus Monkeys of a Recombinant Malaria Vaccine for Plasmodium vivax with a Synthetic Toll-Like Receptor 4 Agonist Formulated in an Emulsion▿‡

Plasmodium vivax is the major cause of malaria outside sub-Saharan Africa and inflicts debilitating morbidity and consequent economic impacts in developing countries. In order to produce a P. vivax vaccine for global use, we have previously reported the development of a novel chimeric recombinant protein, VMP001, based on the circumsporozoite protein (CSP) of P. vivax. Very few adjuvant formulations are currently available for human use. Our interest is to evaluate second-generation vaccine formulations to identify novel combinations of adjuvants capable of inducing strong, long-lasting immune responses. In this study rhesus monkeys were immunized intramuscularly three times with VMP001 in combination with a stable emulsion (SE) or a synthetic Toll-like receptor 4 (TLR4) agonist (glucopyranosyl lipid A [GLA]) in SE (GLA-SE). Sera and peripheral blood mononuclear cells (PBMCs) were tested for the presence of antigen-specific humoral and cellular responses, respectively. All groups of monkeys generated high titers of anti-P. vivax IgG antibodies, as detected by enzyme-linked immunosorbent assays (ELISAs) and immunofluorescence assays. In addition, all groups generated a cellular immune response characterized by antigen-specific CD4+ T cells secreting predominantly interleukin-2 (IL-2) and lesser amounts of tumor necrosis factor (TNF). We conclude that the combination of VMP001 and GLA-SE is safe and immunogenic in monkeys and may serve as a potential second-generation vaccine candidate against P. vivax malaria

Evaluation of in vitro cross-reactivity to avian H5N1 and pandemic H1N1 2009 influenza following prime boost regimens of seasonal influenza vaccination in healthy human subjects: a randomised trial.

Recent studies have demonstrated that inactivated seasonal influenza vaccines (IIV) may elicit production of heterosubtypic antibodies, which can neutralize avian H5N1 virus in a small proportion of subjects. We hypothesized that prime boost regimens of live and inactivated trivalent seasonal influenza vaccines (LAIV and IIV) would enhance production of heterosubtypic immunity and provide evidence of cross-protection against other influenza viruses.In an open-label study, 26 adult volunteers were randomized to receive one of four vaccine regimens containing two doses of 2009-10 seasonal influenza vaccines administered 8 (±1) weeks apart: 2 doses of LAIV; 2 doses of IIV; LAIV then IIV; IIV then LAIV. Humoral immunity assays for avian H5N1, 2009 pandemic H1N1 (pH1N1), and seasonal vaccine strains were performed on blood collected pre-vaccine and 2 and 4 weeks later. The percentage of cytokine-producing T-cells was compared with baseline 14 days after each dose.Subjects receiving IIV had prompt serological responses to vaccine strains. Two subjects receiving heterologous prime boost regimens had enhanced haemagglutination inhibition (HI) and neutralization (NT) titres against pH1N1, and one subject against avian H5N1; all three had pre-existing cross-reactive antibodies detected at baseline. Significantly elevated titres to H5N1 and pH1N1 by neuraminidase inhibition (NI) assay were observed following LAIV-IIV administration. Both vaccines elicited cross-reactive CD4+ T-cell responses to nucleoprotein of avian H5N1 and pH1N1. All regimens were safe and well tolerated.Neither homologous nor heterologous prime boost immunization enhanced serum HI and NT titres to 2009 pH1N1 or avian H5N1 compared to single dose vaccine. However heterologous prime-boost vaccination did lead to in vitro evidence of cross-reactivity by NI; the significance of this finding is unclear. These data support the strategy of administering single dose trivalent seasonal influenza vaccine at the outset of an influenza pandemic while a specific vaccine is being developed.ClinicalTrials.gov NCT01044095